Despite ~10% of the human genome being comprised of secretory proteins, little is known about the physical states of granule lumenal and membrane proteins before and during fusion. This proposal is based upon the hypothesis that the mobility characteristics of granule lumenal contents and of granule membrane proteins shape the secretory response. The proposal will provide fundamental new insights concerning secretory granule structure and function in exocytosis and will provide the first quantitative measures of the rotational and translational mobility of granule lumenal and membrane proteins of individual granules. Our research focuses on events in the exocytotic pathway that occur in the highly specialized domain of the plasma membrane-cytoplasm interface. This region is superbly imaged by total internal reflection fluorescence microscopy (TIRFM), a core technique that we use extensively in our studies. The proposal is supported by strong preliminary results demonstrating distinct mobilities of different lumenal proteins, NPY-Cerulean and tPA-Cerulean. tPA-Cerulean, which has a much lower mobility, is released much more slowly upon fusion of individual granules and the fusion is associated with much slower fusion pore expansion. The rotational and translational mobility of lumenal and granule membrane proteins and of individual granules will be measured by novel combinations of TIRFM, polarization, fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). There are several related goals in the proposal: 1) To understand the MOBILITY CHARACTERISTICS OF PROTEINS IN THE GRANULE LUMEN and reveal their influence in determining the rates of protein and catecholamine release, 2) To determine the translational MOBILITY OF GRANULE MEMBRANE PROTEINS and whether the mobility permits their recruitment by diffusion to the fusion site on the granule membrane, 3) To determine the ROTATIONAL MOBILITY OF ENTIRE GRANULES in order to better define the tethered and/or caged state of the granules before fusion, 4) To determine whether the increase in granule travel immediately before fusion reflects a combination of translational and rotational motion that permits the granule to 'ROLL' into a fusion competent interaction with the plasma membrane, and 5) To measure for the first time the ABSOLUTE DISTANCE BETWEEN THE GRANULE AND THE PLASMA MEMBRANE using supercritical angle emission, thereby determining the timing of engagement of the granule with the plasma membrane before fusion. In addition, it is anticipated that the new techniques will not only be applicable to secretory cell biology, but more generally to the multitude of cell biological issues at the plasma membrane- cytosol interface.